Two-Step In Vitro Model to Evaluate the Cellular Immune Response to SARS-CoV-2.

The cellular immune response plays an important role in COVID-19, caused by SARS-CoV-2. This feature makes use of in vitro models' useful tools to evaluate vaccines and biopharmaceutical effects. Here, we developed a two-step model to evaluate the cellular immune response after SARS-CoV-2 infection-induced or spike protein stimulation in peripheral blood mononuclear cells (PBMC) from both unexposed and COVID-19 (primo-infected) individuals (Step1). Moreover, the supernatants of these cultures were used to evaluate its effects on lung cell lines (A549) (Step2). When PBMC from the unexposed were infected by SARS-CoV-2, cytotoxic natural killer and nonclassical monocytes expressing inflammatory cytokines genes were raised. The supernatant of these cells can induce apoptosis of A549 cells (mock vs. Step2 [mean]: 6.4% × 17.7%). Meanwhile, PBMCs from primo-infected presented their memory CD4+ T cells activated with a high production of IFNG and antiviral genes. Supernatant from past COVID-19 subjects contributed to reduce apoptosis (mock vs. Step2 [ratio]: 7.2 × 1.4) and to elevate the antiviral activity (iNOS) of A549 cells (mock vs. Step2 [mean]: 31.5% × 55.7%). Our findings showed features of immune primary cells and lung cell lines response after SARS-CoV-2 or spike protein stimulation that can be used as an in vitro model to study the immunity effects after SARS-CoV-2 antigen exposure.

Batch chromatography with recycle lag. I-Concept and design.

This is the first of a two-part study in which we explore the concept of batch chromatography with recycle lag, concluding with the design, construction, and experimental validation of a prototype that embodies the physical realization of this concept. Moreover, the apparatus is simple to set up in particular in view of large-scale applications. Here the theory behind batch chromatography with recycle lag is revisited and extended, with emphasis on the mathematical formulation and procedure for deriving the single-column batch analogue of any variant of multicolumn simulated countercurrent chromatography. By resorting to selected examples, namely GE Healthcare Bio-science's three-column periodic countercurrent chromatography, Novasep's sequential multicolumn chromatography, and a few hypothetical multicolumn processes, we discuss how the theory can be operationalized. Finally, we conclude by describing the design of a device or apparatus-an eluate recycling device (ERD)-to physically realize the proposed concept. The ERD implements an approximate "first in, first out" method for organizing and manipulating the to-be-recycled fractions of eluate collected from the chromatography column, where the oldest (first) amount fluid, or 'head' of the fraction, is the first to exit and be recycled to the column.

Batch chromatography with recycle lag. II-Physical realization and experimental validation.

This is the second of a two-part study in which we explore the concept of batch chromatography with recycle lag, concluding with the design, construction, and experimental validation of a prototype-an eluate recycling device (ERD)-that embodies the physical realization of this concept. The ERD implements an approximate "first in, first out" method of organizing and manipulating the to-be-recycled fractions of eluate collected from the chromatography column, where the oldest (first) amount fluid, or 'head' of the fraction, is the first to exit and be recycled back to the column. Moreover, the apparatus is simple to set up in particular in view of large-scale applications. Here we detail the construction of the ERD and assembly of a setup to interconnect the ERD and a chromatography column. Through the coordinated operation of two-way valves and two-position six-port switching valves it is possible to implement a diverse set of configurations or operating modes interconnecting the chromatography column and the ERD. The setup is validated experimentally with success using the separation of a nucleoside mixture by reversed phase chromatography as a model problem. It is also shown that by redesigning the fluid distributor using 3D printing technology the ERD performance can be improved.

Síndrome de hemiconvulsión-hemiplejía-epilepsia: caso clínico y uso de dextrometorfano / Hemiconvulsion-hemiplegia-epilepsy syndrome: case report and use of dextromethorphan

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Predictores pronósticos de desarrollo neurológico en recién nacidos a término con crisis neonatales / Neurodevelopmental outcome predictors of term newborns with neonatal seizures

Introducción: El peso específico de las crisis neonatales en el pronóstico neurológico de recién nacidos a término no se conoce bien, por lo que el objetivo del estudio era describir predictores pronósticos en crisis neonatales. Sujetos y métodos: Estudio observacional prospectivo de recién nacidos a término con crisis clínicas en un centro terciario (2009-2018). Pronóstico adverso se definió como muerte, retraso global del desarrollo, parálisis cerebral o epilepsia. Se analizaron las características perinatales, la etiología, los hallazgos electroencefalográficos, la neuroimagen y los tratamientos antiepilépticos siguiendo un modelo de regresión logística. Resultados: Se incluyó a un total de 102 recién nacidos (52 de los cuales tenían desarrollo normal). Se registraron 12 fallecimientos. En el grupo de supervivientes, 38 niños tuvieron un pronóstico desfavorable (28 con retraso global del desarrollo, 27 con parálisis cerebral, 21 con epilepsia). De las variables pronósticas identificadas en el análisis univariante, las complicaciones perinatales, el inicio de las crisis en el primer día de vida, la actividad basal anormal moderada a grave, un patrón anormal en el electroencefalograma de amplitud integrada y la respuesta al tratamiento continuaron mostrándose como independientemente asociadas a pronóstico adverso después de aplicar un modelo de regresión logística. Conclusiones: Existen datos contradictorios sobre marcadores subrogados en crisis neonatales. Aparte de confirmar el valor predictivo de variables previamente descritas, se halló que la monitorización con electroencefalograma de amplitud integrada constituye una prometedora herramienta diagnóstica. En el futuro, se debería extender su utilización en el abordaje de estos pacientes, lo que sería de vital importancia para un diagnóstico y un tratamiento precoces

Introduction: The concrete burden of neonatal seizures in neurodevelopmental outcome of term newborns is still unknown in literature. The aim of this study was to describe prognostic predictors in neonatal seizures. Subjects and methods: Observational prospective study of term neonates with clinical seizures from a tertiary center (2009-2018). Adverse outcome was determined as death, global developmental delay, cerebral palsy or epilepsy. Perinatal characteristics, etiology, electrographic features, neuroimaging and antiepileptic treatment were analyzed in a logistic regression model. Results: A total of 102 newborns were included (52 infants with normal outcome). Twelve fatalities were registered. In the survival group, 38 children had an adverse outcome (28 global developmental delay, 27 cerebral palsy, 21 epilepsy). From the prognostic variables identified in univariate analysis, perinatal complications, seizure onset in the first day of life, moderate to severe abnormal background activity, abnormal amplitude-integrated EEG pattern, and treatment response remained independently associated with adverse outcome after a logistic regression model. Conclusions: There is conflicting data about surrogate markers in neonatal seizures. Aside from confirming the predictive value of previously described variables, we observed that amplitude-integrated EEG monitoring is a forthcoming prognostic tool. Future approaches may include a wider use of amplitude-integrated EEG monitoring, being crucial for timely seizure identification and prompt treatment

Hydraulic and chemical signalling in the regulation of stomatal conductance and plant water use in field grapevines growing under deficit irrigation.

Effects of irrigation strategies on stomata and plant water use were studied in field-grown grapevines (Vitis vinifera L.). We assessed the importance of root-derived chemical signals vs. hydraulic signalling in stomatal regulation. The experiment included two treatments with the same water added to the soil (50% ETc) applied either to the whole root system (DI) or to half of the roots, alternating irrigation side every 15 days (PRD). Well-watered plants (FI) (100% ETc) and non-irrigated grapevines (NI) were also studied. Partial stomata closure occurred in both PRD and DI plants. [ABA] of xylem sap remained constant during the day and was maintained throughout the season, with higher values in NI plants. Xylem sap pH was not affected by soil water availability. A positive correlation between ψpd and maximum g s was found, indicating that grapevine stomata strongly respond to plant water status. In contrast, ABA did not explain stomatal control at veraison. At mid-ripening g s was significantly correlated with ABA, apparently interacting with the rise in xylem sap pH. Therefore, our data suggest that hydraulic feedback and feed-forward root-to-shoot chemical signalling mechanisms might be involved in the control of stomata in response to decreased soil water availability, hydraulic signals playing the dominant role.

Impact of deficit irrigation on water use efficiency and carbon isotope composition (delta13C) of field-grown grapevines under Mediterranean climate.

The objective of this study was to evaluate the effect of deficit irrigation on intrinsic water use efficiency (A/g(s)) and carbon isotope composition (delta13C) of two grapevine cultivars (Moscatel and Castelão), growing in a commercial vineyard in SW Portugal. The study was done in two consecutive years (2001 and 2002). The treatments were full irrigation (FI), corresponding to 100% of crop evapotranspiration (ETc), rain-fed (no irrigation, NI), and two types of deficit irrigation (50% ETc): (i) by supplying the water either to one side of the root system or to the other, which is partial rootzone drying (PRD), or (ii) dividing the same amount of water by the two sides of the root system, the normal deficit irrigation (DI). The water supplied to the PRD treatment alternated sides approximately every 15 d. The values of predawn leaf water potential (Psi(pd)) and the cumulative integral of Psi(pd) (S(Psi)) during the season were lower in 2001 than in the 2002 growing season. Whereas differences in Psi(pd) and S(Psi) between PRD and DI were not significantly different in 2001, in 2002 (a dryer year) both cultivars showed lower values of S(Psi) in the PRD treatment as compared with the DI treatment. This suggests that partial rootzone drying may have a positive effect on water use under dryer conditions, either as a result of better stomatal control and/or reduced vigour. The effects of the water treatments on delta13C were more pronounced in whole grape berries and pulp than in leaves. The delta13C of pulp showed the best correlation with intrinsic water use efficiency (A/g(s)) as well as with S(Psi). In spite of the better water status observed in PRD compared with DI in the two cultivars in 2002, no statistical differences between the two treatments were observed in A/g(s) and delta13C. On the other hand, they showed a higher delta13C compared with FI. In conclusion, it is apparent that the response to deficit irrigation varies with the environmental conditions of the particular year, the driest conditions exacerbating the differences among treatments. The highest values of delta13C found in the pulp of NI vines in Castelão compared with Moscatel suggest different sensitivities to water deficits in the two cultivars, as was empirically observed.

Partial rootzone drying: regulation of stomatal aperture and carbon assimilation in field-grown grapevines (Vitis vinifera cv. Moscatel).

The effects of 'partial rootzone drying' (PRD) irrigation compared with other irrigation systems, namely non-irrigated (NI), full irrigation (FI) and deficit irrigation (DI), on stomatal conductance and carbon assimilation were evaluated in field-grown grapevines (Vitis vinifera L. cv. Moscatel). At the end of the growing season, pre-dawn leaf water potential was highest in FI (-0.18 ± 0.01 MPa; mean ± s.e.), intermediate in PRD (-0.30± 0.01 MPa) and DI (-0.36 ± 0.02 MPa), and lowest in NI vines (-0.64 ± 0.03 MPa). Stomatal conductance measured under controlled conditions of light and temperature was reduced in NI (ca 60%) and PRD (ca 30%) vines compared with DI and FI vines. Under ambient conditions, NI vines had lower rates of stomatal conductance (ca26%), net CO2 assimilation (ca 28%) and light-adapted PSII quantum yields (ca 47%) than PRD, DI and FI vines. No significant differences were found among the three irrigated treatments. Both maximum electron transport rate (Jmax; ca 30%) and triose-phosphate utilization rates (TPU; ca 20%) were significantly lower in NI and PRD vines than in DI and FI vines. Carbon isotope composition (δ13C) of grape berries was highest in NI vines (-24.3), followed by PRD (-25.4) and DI (-25.8) and lowest in FI (-26.4) vines, suggesting a long-term increase in the efficiency of leaf gas exchange in NI compared with PRD, DI and FI vines. Sap-flow data and estimates of relative stomatal limitation are in accordance with the observed stomatal closure in PRD vines. In this study, we show that PRD irrigation was able to maintain a vine water status closed to FI, but with double water use efficiency, which was due to a reduction of stomatal conductance with no significant decrease in carbon assimilation.

Partial rootzone drying: effects on growth and fruit quality of field-grown grapevines (Vitis vinifera).

A study to assess the effects of the Partial Rootzone Drying (PRD) irrigation strategy in comparison to other irrigation systems was carried out in southern Portugal in two field-grown grapevines varieties, Moscatel and Castelão. We addressed the question of whether by regulating growth and plant water use, the PRD system would enable an equilibrated vegetative development, leading to a favourable capture of solar radiation for photoassimilate production and, at the same time to provide an optimum environment for fruit maturation. Three irrigation schemes were applied in addition to the non-irrigated (NI) vines: partial root drying (PRD), 50% of crop evapotranspiration (ETc), supplied to only one side of the root system while the other one was allowed to dry, alternating sides every 15 days; deficit irrigated (DI), 50% ETc supplied, half to each side of the root system and full irrigated (FI, 100% ETc). During the whole season FI plants of both varieties exhibited a high leaf predawn water potential (ψpd , ca-0.2MPa) while a progressive decline was observed in NI plants, reaching ψpd values near -0.7 MPa at the end of August. PRD and DI presented intermediate values. PRD vines exhibited a stronger control over vegetative growth as compared with DI and FI plants. This was expressed by lower values of total leaf area at harvest, leaf layer number, canopy wideness and water shoots number, allowing a higher light interception at the cluster zone that induced an improvement in some berry quality characteristics. Watering had no significant effects on sugar accumulation in the berries but led to a favourable increase in the must titratable acidity, mainly in Castelão. Whereas in DI and FI treatments berry skin anthocyanins and phenols content were always lower than in NI, in PRD there was either no reduction or the reduction was much lower than in the other irrigation treatments. Water use efficiency (WUE) was increased by about 80% in PRD and DI when compared with FI, as a result of almost similar yields in the three treatments. Yield gains of irrigated plants in relation to NI were modest, explained by the rainy spring in both years.